The toxic gases O3, NO2 and cigarette smoke (CS) cause lung damage. The cellular and molecular mechanisms of the damage are complex, but oxidant reactions are thought to play a major role. The respiratory tract epithelial lining fluids (RTLFs) are among the first biological systems to come into contact with inhaled gases. RTLFs contain many different antioxidants, including mucin, uric acid, alpha-tocopherol, ascorbic acid, and glutathione. Although much is known about the reactions of individual isolated antioxidants with oxidants, very little is known about the integrated antioxidant defenses of RTLFs from different parts of the respiratory tract. RTLFs may not only protect against oxidative injury to underlying cells, but oxidation of their constituents may generate cytotoxic products. In addition, several of their protein and lipid constituents have specific functions, such as elastase-inhibiting capacity and surface tension-lowering ability. The applicants will apply a strategy in which temporal losses of different antioxidants are quantitated in relation to the onset of structural and functional protein and lipid damage in RTLFs exposed to O3, NO2 and CS. The cellular effects of oxidized RTLFs will also be examined. The specific aims are: (i) to identify the antioxidants present in RTLFs from different parts of the respiratory tract; (ii) to identify the most important antioxidants in RTLFs that can protect the proteins and lipids present against damage by O3, NO2 and CS, singly or in combination; (iii) to study the effects of oxidation of RTLF components upon their cytoxicity and their biochemical functions. Highly- sensitive, specific assays will be used to measure antioxidants, lipid peroxidation and oxidative damage to proteins. Understanding the integrated protective role of antioxidants in RTLFs may well provide new insights into the sequence of biochemical and cellular events occurring in the respiratory tract after exposure to toxic gases. This may lead to the development of strategies for protective therapeutic interventions and of "biomarkers" for O3-induced damage.